Deficiency of factor-inhibiting HIF creates a tumor-promoting immune microenvironment.

Hypoxia signaling influences tumor development through both cell-intrinsic and -extrinsic pathways. Inhibiting hypoxia-inducible factor (HIF) function has recently been approved as a cancer treatment strategy. Hence, it is important to understand how regulators of HIF may affect tumor growth under physiological conditions. Here we report that in aging mice factor-inhibiting HIF (FIH), one of the most studied negative regulators of HIF, is a haploinsufficient suppressor of spontaneous B cell lymphomas, particular pulmonary B cell lymphomas. FIH deficiency alters immune composition in aged mice and creates a tumor-supportive immune environment demonstrated in syngeneic mouse tumor models. Mechanistically, FIH-defective myeloid cells acquire tumor-supportive properties in response to signals secreted by cancer cells or produced in the tumor microenvironment with enhanced arginase expression and cytokine-directed migration. Together, these data demonstrate that under physiological conditions, FIH plays a key role in maintaining immune homeostasis and can suppress tumorigenesis through a cell-extrinsic pathway.

5-Substituted Pyridine-2,4-dicarboxylate Derivatives Have Potential for Selective Inhibition of Human Jumonji-C Domain-Containing Protein 5.

Jumonji-C domain-containing protein 5 (JMJD5) is a 2-oxoglutarate (2OG)-dependent oxygenase that plays important roles in development, circadian rhythm, and cancer through unclear mechanisms. JMJD5 has been reported to have activity as a histone protease, as an Nε-methyl lysine demethylase, and as an arginine residue hydroxylase. Small-molecule JMJD5-selective inhibitors will be useful for investigating its (patho)physiological roles. Following the observation that the broad-spectrum 2OG oxygenase inhibitor pyridine-2,4-dicarboxylic acid (2,4-PDCA) is a 2OG-competing JMJD5 inhibitor, we report that 5-aminoalkyl-substituted 2,4-PDCA derivatives are potent JMJD5 inhibitors manifesting selectivity for JMJD5 over other human 2OG oxygenases. Crystallographic analyses with five inhibitors imply induced fit binding and reveal that the 2,4-PDCA C5 substituent orients into the JMJD5 substrate-binding pocket. Cellular studies indicate that the lead compounds display similar phenotypes as reported for clinically observed JMJD5 variants, which have a reduced catalytic activity compared to wild-type JMJD5.

First-in-Class Inhibitors of the Ribosomal Oxygenase MINA53.

MINA53 is a JmjC domain 2-oxoglutarate-dependent oxygenase that catalyzes ribosomal hydroxylation and is a target of the oncogenic transcription factor c-MYC. Despite its anticancer target potential, no small-molecule MINA53 inhibitors are reported. Using ribosomal substrate fragments, we developed mass spectrometry assays for MINA53 and the related oxygenase NO66. These assays enabled the identification of 2-(aryl)alkylthio-3,4-dihydro-4-oxoypyrimidine-5-carboxylic acids as potent MINA53 inhibitors, with selectivity over NO66 and other JmjC oxygenases. Crystallographic studies with the JmjC demethylase KDM5B revealed active site binding but without direct metal chelation; however, molecular modeling investigations indicated that the inhibitors bind to MINA53 by directly interacting with the iron cofactor. The MINA53 inhibitors manifest evidence for target engagement and selectivity for MINA53 over KDM4-6. The MINA53 inhibitors show antiproliferative activity with solid cancer lines and sensitize cancer cells to conventional chemotherapy, suggesting that further work investigating their potential in combination therapies is warranted.

Developmentally regulated GTPases: structure, function and roles in disease.

GTPases are a large superfamily of evolutionarily conserved proteins involved in a variety of fundamental cellular processes. The developmentally regulated GTP-binding protein (DRG) subfamily of GTPases consists of two highly conserved paralogs, DRG1 and DRG2, both of which have been implicated in the regulation of cell proliferation, translation and microtubules. Furthermore, DRG1 and 2 proteins both have a conserved binding partner, DRG family regulatory protein 1 and 2 (DFRP1 and DFRP2), respectively, that prevents them from being degraded. Similar to DRGs, the DFRP proteins have also been studied in the context of cell growth control and translation. Despite these proteins having been implicated in several fundamental cellular processes they remain relatively poorly characterized, however. In this review, we provide an overview of the structural biology and biochemistry of DRG GTPases and discuss current understanding of DRGs and DFRPs in normal physiology, as well as their emerging roles in diseases such as cancer.

Human 2-oxoglutarate-dependent oxygenases: nutrient sensors, stress responders, and disease mediators.

Fe(II)/2-oxoglutarate (2OG)-dependent oxygenases are a conserved enzyme class that catalyse diverse oxidative reactions across nature. In humans, these enzymes hydroxylate a broad range of biological substrates including DNA, RNA, proteins and some metabolic intermediates. Correspondingly, members of the 2OG-dependent oxygenase superfamily have been linked to fundamental biological processes, and found dysregulated in numerous human diseases. Such findings have stimulated efforts to understand both the biochemical activities and cellular functions of these enzymes, as many have been poorly studied. In this review, we focus on human 2OG-dependent oxygenases catalysing the hydroxylation of protein and polynucleotide substrates. We discuss their modulation by changes in the cellular microenvironment, particularly with respect to oxygen, iron, 2OG and the effects of oncometabolites. We also describe emerging evidence that these enzymes are responsive to cellular stresses including hypoxia and DNA damage. Moreover, we examine how dysregulation of 2OG-dependent oxygenases is associated with human disease, and the apparent paradoxical role for some of these enzymes during cancer development. Finally, we discuss some of the challenges associated with assigning biochemical activities and cellular functions to 2OG-dependent oxygenases.

Hypoxia drives glucose transporter 3 expression through hypoxia-inducible transcription factor (HIF)-mediated induction of the long noncoding RNA NICI.

Hypoxia-inducible transcription factors (HIFs) directly dictate the expression of multiple RNA species including novel and as yet uncharacterized long noncoding transcripts with unknown function. We used pan-genomic HIF-binding and transcriptomic data to identify a novel long noncoding RNA Noncoding Intergenic Co-Induced transcript (NICI) on chromosome 12p13.31 which is regulated by hypoxia via HIF-1 promoter-binding in multiple cell types. CRISPR/Cas9-mediated deletion of the hypoxia-response element revealed co-regulation of NICI and the neighboring protein-coding gene, solute carrier family 2 member 3 (SLC2A3) which encodes the high-affinity glucose transporter 3 (GLUT3). Knockdown or knockout of NICI attenuated hypoxic induction of SLC2A3, indicating a direct regulatory role of NICI in SLC2A3 expression, which was further evidenced by CRISPR/Cas9-VPR-mediated activation of NICI expression. We also demonstrate that regulation of SLC2A3 is mediated through transcriptional activation rather than posttranscriptional mechanisms because knockout of NICI leads to reduced recruitment of RNA polymerase 2 to the SLC2A3 promoter. Consistent with this we observe NICI-dependent regulation of glucose consumption and cell proliferation. Furthermore, NICI expression is regulated by the von Hippel-Lindau (VHL) tumor suppressor and is highly expressed in clear cell renal cell carcinoma (ccRCC), where SLC2A3 expression is associated with patient prognosis, implying an important role for the HIF/NICI/SLC2A3 axis in this malignancy.

Myc-induced nuclear antigen constrains a latent intestinal epithelial cell-intrinsic anthelmintic pathway.

Expulsion of parasitic gastrointestinal nematodes requires diverse effector mechanisms coordinated by a Th2-type response. The evolutionarily conserved JmjC protein; Myc Induced Nuclear Antigen (Mina) has been shown to repress IL4, a key Th2 cytokine, suggesting Mina may negatively regulate nematode expulsion. Here we report that expulsion of the parasitic nematode Trichuris muris was indeed accelerated in Mina deficient mice. Unexpectedly, this was associated not with an elevated Th2- but rather an impaired Th1-type response. Further reciprocal bone marrow chimera and conditional KO experiments demonstrated that retarded parasite expulsion and a normal Th1-type response both required Mina in intestinal epithelial cells (IECs). Transcriptional profiling experiments in IECs revealed anti-microbial α-defensin peptides to be the major target of Mina-dependent retention of worms in infected mice. In vitro exposure to recombinant α-defensin peptides caused cytotoxic damage to whipworms. These results identify a latent IEC-intrinsic anthelmintic pathway actively constrained by Mina and point to α-defensins as important effectors that together with Mina may be attractive therapeutic targets for the control of nematode infection.

The emerging roles of ribosomal histidyl hydroxylases in cell biology, physiology and disease.

Hydroxylation is a novel protein modification catalyzed by a family of oxygenases that depend on fundamental nutrients and metabolites for activity. Protein hydroxylases have been implicated in a variety of key cellular processes that play important roles in both normal homeostasis and pathogenesis. Here, in this review, we summarize the current literature on a highly conserved sub-family of oxygenases that catalyze protein histidyl hydroxylation. We discuss the evidence supporting the biochemical assignment of these emerging enzymes as ribosomal protein hydroxylases, and provide an overview of their role in immunology, bone development, and cancer.

OH, the Places You'll Go! Hydroxylation, Gene Expression, and Cancer.

Hydroxylation is an emerging modification generally catalyzed by a family of ∼70 enzymes that are dependent on oxygen, Fe(II), ascorbate, and the Kreb's cycle intermediate 2-oxoglutarate (2OG). These "2OG oxygenases" sit at the intersection of nutrient availability and metabolism where they have the potential to regulate gene expression and growth in response to changes in co-factor abundance. Characterized 2OG oxygenases regulate fundamental cellular processes by catalyzing the hydroxylation or demethylation (via hydroxylation) of DNA, RNA, or protein. As such they have been implicated in various syndromes and diseases, but particularly cancer. In this review we discuss the emerging role of 2OG oxygenases in gene expression control, examine the regulation of these unique enzymes by nutrient availability and metabolic intermediates, and describe these properties in relation to the expanding role of these enzymes in cancer.

Optimal translational termination requires C4 lysyl hydroxylation of eRF1.

Efficient stop codon recognition and peptidyl-tRNA hydrolysis are essential in order to terminate translational elongation and maintain protein sequence fidelity. Eukaryotic translational termination is mediated by a release factor complex that includes eukaryotic release factor 1 (eRF1) and eRF3. The N terminus of eRF1 contains highly conserved sequence motifs that couple stop codon recognition at the ribosomal A site to peptidyl-tRNA hydrolysis. We reveal that Jumonji domain-containing 4 (Jmjd4), a 2-oxoglutarate- and Fe(II)-dependent oxygenase, catalyzes carbon 4 (C4) lysyl hydroxylation of eRF1. This posttranslational modification takes place at an invariant lysine within the eRF1 NIKS motif and is required for optimal translational termination efficiency. These findings further highlight the role of 2-oxoglutarate/Fe(II) oxygenases in fundamental cellular processes and provide additional evidence that ensuring fidelity of protein translation is a major role of hydroxylation.

Dynamic regulatory network controlling TH17 cell differentiation.

Despite their importance, the molecular circuits that control the differentiation of naive T cells remain largely unknown. Recent studies that reconstructed regulatory networks in mammalian cells have focused on short-term responses and relied on perturbation-based approaches that cannot be readily applied to primary T cells. Here we combine transcriptional profiling at high temporal resolution, novel computational algorithms, and innovative nanowire-based perturbation tools to systematically derive and experimentally validate a model of the dynamic regulatory network that controls the differentiation of mouse TH17 cells, a proinflammatory T-cell subset that has been implicated in the pathogenesis of multiple autoimmune diseases. The TH17 transcriptional network consists of two self-reinforcing, but mutually antagonistic, modules, with 12 novel regulators, the coupled action of which may be essential for maintaining the balance between TH17 and other CD4(+) T-cell subsets. Our study identifies and validates 39 regulatory factors, embeds them within a comprehensive temporal network and reveals its organizational principles; it also highlights novel drug targets for controlling TH17 cell differentiation.

The hypoxia-inducible transcription factor pathway regulates oxygen sensing in the simplest animal, Trichoplax adhaerens.

The hypoxic response in humans is mediated by the hypoxia-inducible transcription factor (HIF), for which prolyl hydroxylases (PHDs) act as oxygen-sensing components. The evolutionary origins of the HIF system have been previously unclear. We demonstrate a functional HIF system in the simplest animal, Trichoplax adhaerens: HIF targets in T. adhaerens include glycolytic and metabolic enzymes, suggesting a role for HIF in the adaptation of basal multicellular animals to fluctuating oxygen levels. Characterization of the T. adhaerens PHDs and cross-species complementation assays reveal a conserved oxygen-sensing mechanism. Cross-genomic analyses rationalize the relative importance of HIF system components, and imply that the HIF system is likely to be present in all animals, but is unique to this kingdom.

PHF8, a gene associated with cleft lip/palate and mental retardation, encodes for an Nepsilon-dimethyl lysine demethylase.

Mutations of human PHF8 cluster within its JmjC encoding exons and are linked to mental retardation (MR) and a cleft lip/palate phenotype. Sequence comparisons, employing structural insights, suggest that PHF8 contains the double stranded beta-helix fold and ferrous iron binding residues that are present in 2-oxoglutarate-dependent oxygenases. We report that recombinant PHF8 is an Fe(II) and 2-oxoglutarate-dependent N(epsilon)-methyl lysine demethylase, which acts on histone substrates. PHF8 is selective in vitro for N(epsilon)-di- and mono-methylated lysine residues and does not accept trimethyl substrates. Clinically observed mutations to the PHF8 gene cluster in exons encoding for the double stranded beta-helix fold and will therefore disrupt catalytic activity. The PHF8 missense mutation c.836C>T is associated with mild MR, mild dysmorphic features, and either unilateral or bilateral cleft lip and cleft palate in two male siblings. This mutant encodes a F279S variant of PHF8 that modifies a conserved hydrophobic region; assays with both peptides and intact histones reveal this variant to be catalytically inactive. The dependence of PHF8 activity on oxygen availability is interesting because the occurrence of fetal cleft lip has been demonstrated to increase with maternal hypoxia in mouse studies. Cleft lip and other congenital anomalies are also linked indirectly to maternal hypoxia in humans, including from maternal smoking and maternal anti-hypertensive treatment. Our results will enable further studies aimed at defining the molecular links between developmental changes in histone methylation status, congenital disorders and MR.

Hypoxia, hypoxia-inducible factors (HIF), HIF hydroxylases and oxygen sensing.

This article outlines the need for a homeostatic response to alterations in cellular oxygenation. It describes work on erythropoietin control that led to the discovery of the hypoxia-inducible transcription factor (HIF-1) and the parallel recognition that this system was responsive to a widespread oxygen-sensing mechanism. Subsequently, multiple HIF isoforms have been shown to have overlapping but non-redundant functions, controlling expression of genes involved in diverse processes such as angiogenesis, vascular tone, metal transport, glycolysis, mitochondrial function, cell growth and survival. The major role of prolyl and asparaginyl hydroxylation in regulating HIFs is described, as well as the identification of PHD1-3 and FIH as the oxygen-sensing enzymes responsible for these hydroxylations. Current understanding of other processes that modulate overall HIF activity, including influences from other signalling mechanisms such as kinases and nitric oxide levels, and the existence of a variety of feedback loops are outlined. The effects of some mutations in this pathway are documented as is knowledge of other substrates for these enzymes. The importance of PHD1-3 and FIH, and the large family of 2-oxoglutarate and iron(II)-dependent dioxygenases of which they are a part, in biology and medicine are discussed.

Sprouty2 association with B-Raf is regulated by phosphorylation and kinase conformation.

Sprouty2 is a feedback regulator that controls the Ras/Raf/MEK/extracellular signal-regulated kinase mitogen-activated protein kinase (MAPK) pathway at multiple levels, one way being through direct interaction with Raf kinases. Consistent with a role as a tumor suppressor, Sprouty2 expression is often down-regulated in human cancers. However, Sprouty2 is up-regulated in some cancers, suggesting the existence of posttranscriptional mechanisms that permit evasion of Sprouty2-mediated antitumorigenic properties. We report that MAPK activation induces Sprouty2 phosphorylation on six serine residues, which reduced Sprouty2 association with wild-type B-Raf. Mutation of these six serines to nonphosphorylatable alanines increased the ability of Sprouty2 to inhibit growth factor-induced MAPK activation. Oncogenic B-Raf mutants such as B-Raf V600E did not associate with Sprouty2, but this resistance to Sprouty2 binding was not due to phosphorylation. Instead, the active kinase conformation induced by oncogenic mutation prevents Sprouty2 binding. These results reveal a dual mechanism that affects the Sprouty2/B-Raf interaction: Sprouty phosphorylation and B-Raf conformation.

MYPT1, the targeting subunit of smooth-muscle myosin phosphatase, is a substrate for the asparaginyl hydroxylase factor inhibiting hypoxia-inducible factor (FIH).

The asparaginyl hydroxylase FIH [factor inhibiting HIF (hypoxia-inducible factor)] was first identified as a protein that inhibits transcriptional activation by HIF, through hydroxylation of an asparagine residue in the CAD (C-terminal activation domain). More recently, several ARD [AR (ankyrin repeat) domain]-containing proteins were identified as FIH substrates using FIH interaction assays. Although the function(s) of these ARD hydroxylations is unclear, expression of the ARD protein Notch1 was shown to compete efficiently with HIF CAD for asparagine hydroxylation and thus to enhance HIF activity. The ARD is a common protein domain with over 300 examples in the human proteome. However, the extent of hydroxylation among ARD proteins, and the ability of other members to compete with HIF-CAD for FIH, is not known. In the present study we assay for asparagine hydroxylation in a bioinformatically predicted FIH substrate, the targeting subunit of myosin phosphatase, MYPT1. Our results confirm hydroxylation both in cultured cells and in endogenous protein purified from animal tissue. We show that the extent of hydroxylation at three sites is dependent on FIH expression level and that hydroxylation is incomplete under basal conditions even in the animal tissue. We also show that expression of MYPT1 enhances HIF-CAD activity in a manner consistent with competition for FIH and that this property extends to other ARD proteins. These results extend the range of FIH substrates and suggest that cross-competition between ARDs and HIF-CAD, and between ARDs themselves, may be extensive and have important effects on hypoxia signalling.

Oxygen sensing and hypoxia-induced responses.

Low cellular oxygenation (hypoxia) represents a significant threat to the viability of affected tissues. Multicellular organisms have evolved a highly conserved signalling pathway that directs many of the changes in gene expression that underpin physiological oxygen homoeostasis. Oxygen-sensing enzymes in this pathway control the activity of the HIF (hypoxia-inducible factor) transcription factor by the direct incorporation of molecular oxygen into the post-translational hydroxylation of specific residues. This represents the canonical hypoxia signalling pathway which regulates a plethora of genes involved in adaptation to hypoxia. The HIF hydroxylases have been identified in other biological contexts, consistent with the possibility that they have other substrates. Furthermore, several intracellular proteins have been demonstrated, directly or indirectly, to be hydroxylated, although the protein hydroxylases responsible have yet to be identified. This chapter will summarize what is currently known about the canonical HIF hydroxylase signalling pathway and will speculate on the existence of other oxygen-sensing enzymes and the role they may play in signalling hypoxia through other pathways.

Actin-myosin-based contraction is responsible for apoptotic nuclear disintegration.

Membrane blebbing during the apoptotic execution phase results from caspase-mediated cleavage and activation of ROCK I. Here, we show that ROCK activity, myosin light chain (MLC) phosphorylation, MLC ATPase activity, and an intact actin cytoskeleton, but not microtubular cytoskeleton, are required for disruption of nuclear integrity during apoptosis. Inhibition of ROCK or MLC ATPase activity, which protect apoptotic nuclear integrity, does not affect caspase-mediated degradation of nuclear proteins such as lamins A, B1, or C. The conditional activation of ROCK I was sufficient to tear apart nuclei in lamin A/C null fibroblasts, but not in wild-type fibroblasts. Thus, apoptotic nuclear disintegration requires actin-myosin contractile force and lamin proteolysis, making apoptosis analogous to, but distinct from, mitosis where nuclear disintegration results from microtubule-based forces and from lamin phosphorylation and depolymerization.

SPRY2 is an inhibitor of the ras/extracellular signal-regulated kinase pathway in melanocytes and melanoma cells with wild-type BRAF but not with the V599E mutant.

BRAF mutations result in constitutively active BRAF kinase activity and increased extracellular signal-regulated kinase (ERK) signaling and cell proliferation. Initial studies have shown that BRAF mutations occur at a high frequency in melanocytic nevi and metastatic lesions, but recent data have revealed much lower incidence of these mutations in early-stage melanoma, implying that other factors may contribute to melanoma pathogenesis in a wild-type (WT) BRAF context. To identify such contributing factors, we used microarray gene expression profiling to screen for differences in gene expression between a panel of melanocytic and melanoma cell lines with WT BRAF and a group of melanoma cell lines with the V599E BRAF mutation. We found that SPRY2, an inhibitor homologous to SPRY4, which was previously shown to suppress Ras/ERK signaling via direct binding to Raf-1, had reduced expression in WT BRAF cells. Using small interfering RNA-mediated SPRY2 knockdown, we showed that SPRY2 acts as an inhibitor of ERK signaling in melanocytes and WT BRAF melanoma cells, but not in cell lines with the V599E mutation. We also show that SPRY2 and SPRY4 directly bind WT BRAF but not the V599E and other exon 15 BRAF mutants. These data suggest that SPRY2, an inhibitor of ERK signaling, may be bypassed in melanoma cells either by down-regulation of its expression in WT BRAF cells, or by the presence of the BRAF mutation.